Cathepsin H purified from porcine spleens was studied for its specificity against various peptide and denatured protein substrates. The enzyme degraded all peptide substrates exclusively by an aminopeptidase activity. The enzyme preferentially released NH2-terminal amino acid residues with large hydrophobic (Phe, Trp, Leu, and Tyr) or basic (Arg and Lys) side chains. Amino acids containing small or polar side chains were not released. Peptides with a proline in the NH2-terminal or penultimate positions were not hydrolyzed either. Large polypeptides such as reduced and carboxymethylated soybean trypsin inhibitor and aldolase were not degraded. These results indicate that cathepsin H is an exopeptidase but not an endopeptidase. We propose that the biological role of this enzyme is the degradation of tissue proteins in lysosomes by its aminopeptidase activity.